Community-acquired pneumonia (CAP) is a leading cause of death among the elderly (>65 years);despite access to antimicrobials and supportive critical care, the case-fatality rate for elderly with CAP is 15-30%. Toll- like receptors (TLR) are pattern recognition molecules that identify molecular patterns associated with invading microorganisms and initiate the innate immune response (e.g. NFkB activation, cytokine production). It is now evident that aging is associated with TLR dysfunction;phagocytes isolated from aged animals exhibit decreased TLR expression, impaired TLR signaling, and a reduction in TLR-mediated cytokine production. Currently the impact of age-dependent TLR dysfunction (ADTD) in the lungs is unknown. However, ADTD may help explain: 1) why CAP is more severe in the elderly;2) why 10% of the elderly with pneumonia show no overt clinical signs of infection such as fever, cough, or dyspnea;and 3) why lack of fever is a prognostic indicator of poor clinical outcome. We hypothesize that ADTD occurs in lungs of aged animals and that it increases susceptibility to pneumonia. Laboratory observations that support this hypothesis include: 1) decreased TLR-1, -2, and -4 protein levels in the lungs of aged mice versus young mice;2) aged mice infected with Streptococcus pneumoniae develop fulminate pneumonia and die within 36 hours whereas young mice remain relatively healthy;3) diminished NFkB activation in the lungs of aged mice during infection;and 4) reduced TNF1 and IL-6 levels in the lungs of aged mice following tracheal challenge with S. pneumoniae components. The focus of this proposal is ADTD in the lungs during pneumonia. We will: Aim 1: Determine the effects of aging on TLRs and TLR signaling protein levels. Using Real Time (RT)- PCR, quantitative Western blot, and FACs analysis we will determine if mRNA and protein levels of TLRs 1-9, and the TLR cell signaling molecules Myd88, IRAK-1, and IRAK-4 are decreased in alveolar macrophages and type II pneumocytes (lung mucosal epithelial cells) isolated from aged versus young and mature mice. Aim 2: Determine the effects of aging on the TLR response to S. pneumoniae components in the lung. Using primary lung cells and live mice, we will determine the effects of aging on phosphorylation of the TLR cell signaling protein IRAK-1, NFkB activation, cytokine production, and expression of leukocyte adhesion molecules in response to peptidoglycan and pneumolysin, TLR-2 and TLR-4 ligands, respectively. -Completion of this proposal will expand our understanding of ADTD to include alveolar macrophages and mucosal epithelial cells, the primary point of contact for respiratory tract pathogens in the lungs. Proposed experiments may determine that ADTD is a principal mechanism responsible for the increased susceptibility of the elderly to pneumonia. This project meets established NIA goals of examining the effects of age-related changes on functional disease outcomes. PUBLIC HEALTH RELEVANCE: Toll-like receptors (TLR) recognize invading microorganisms and initiate the host innate immune response. Evidence collected in our laboratory indicates that age-dependent TLR dysfunction (ADTD) occurs in the lungs. This proposal will determine if alveolar macrophages and mucosal epithelial cells, the primary point of contact for respiratory tract pathogens in the lungs, experience ADTD.